Diagnostic compositions and devices utilizing same

ABSTRACT

A dry chemistry reagent matrix composition is provided containing a matrix material and a reagent composition containing 3-methyl-6-(sulfonate salt)-benzothiazolinone-(2)-hydrazone (MBTH-S), N-ethyl-N-(3-sulfopropyl)aniline, and an oxidase enzyme or a peroxidase enzyme or a mixture thereof. The dry chemistry reagent matrix composition is useful in reagent test strips for determining the presence or concentration of an analyte in a fluid sample, such as blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part (CIP) of U.S. Ser. No.09/037,653, filed Mar. 10, 1998, now U.S. Pat. No. 5,885,790 whichapplication is a continuation of U.S. Ser. No. 08/628,794, filed Apr. 5,1996, now U.S. Pat. No. 5,776,719. These patent applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to dye compositions used for colorimetricdetermination of a chemical or biochemical component (analyte) in anaqueous fluid, such as blood. In particular, the present inventionrelates to the field of dry reagent matrix compositions for use on teststrips adapted to receive a liquid sample of body fluid whereby a dyeindicator composition wetted by the fluid reacts with an analyte in thebody fluid and provides a visual or color indication of the presence orconcentration of the analyte.

2. State of the Art

Indicator compositions for use in devices for color indication ofvarious analytes in liquid samples, such as body fluids, are well knownin the art and are embodied in numerous commercial products, such as dryreagent test strips. In such test strips, a dye or dye couplecomposition is typically formulated in a solution which is applied to atest strip matrix and then dried to form a dry chemistry reagent systemon the test strip. The dry chemistry reagent system commonly involves anoxidizable dye or dye couple composition in combination with a oxidaseor peroxidase specific for the analyte to be tested. When the drychemistry reagent system is contacted with a liquid sample containingthe analyte, the analyte reacts with its corresponding oxidase orperoxidase producing hydrogen peroxide which in turn oxidizes the dye ordye couple to produce the desired color change thereby indicating thepresence or concentration of the analyte.

Example of such dry chemistry reagent systems are disclosed by Phillipset al. in U.S. Pat. Nos. 4,734,360; 5,059,394 and 5,304,468; by Yu inU.S. Pat. No. 5,453,360; Hoenes in U.S. Pat. No. 5,334,508 and byHochstrasser in U.S. Pat. Nos. 3,964,871 and 4,059,407. The disclosuresof these patents are incorporated herein by reference in their entirety.

A number of dry chemistry reagent systems have been incorporated intovarious commercial products. While such products generally provideacceptable indication and testing under some conditions, certainproblems exist with prior dry chemistry systems. For example, in somedry chemistry reagent systems, the dye components tend to sublime overtime from the test strip so that when the test strip is used by theconsumer it may not provide an accurate indication. This problem limitsthe shelf life of the test strips.

Additionally, in some dry chemistry reagent systems, the color changeprovided by the dye or dye couple components continues to change overtime rather than reaching a stable end point. When such dye systems areemployed, the color indication must be accurately read at specific timeintervals in order to obtain an accurate indication of the presence orconcentration of an analyte.

Moreover, some dry chemistry reagent systems require a low pH to providethe necessary stability for the dye or dye couple system. However, thepH of such systems is often below the desired pH level for stability ofthe enzyme used in the dry chemistry system. Therefore, the amount ofenzyme employed in the formulation must be increased to compensate forthe enzyme's instability, thus increasing the cost of the dry chemistryreagent system. Additionally, when the enzyme is used under non-optimalpH conditions, the enzyme may give undesired or false indications of thepresence or concentration of the analyte.

Finally, in some dye or dye couple systems, the optimum wavelength fordetermining the presence or concentration of an analyte is oftenobscured or interfered with by components of the body fluid beinganalyzed, such as hemoglobin.

In view of the above, it is an object of this invention to provide a drychemistry reagent system having improved stability regarding sublimationof the dye or dye couple system from the dry chemistry test strips.

It is also an object of this invention to provide a dry chemistryreagent system which rapidly produces a stable end point in a shortperiod of time thus eliminating the time dependent measurements ordeterminations by the user.

It is a further object of this invention to provide a dry chemistryreagent system which can be formulated and used at a pH range moreclosely approximating physiological pH thus providing a more stablesystem for the enzyme or enzymes employed in the manufacture and use ofthe dry chemistry system.

It is a still further object of this invention to provide a drychemistry reagent system which permits an accurate determination of thepresence or concentration of an analyte in a body fluid withoutsignificant spectral interference by components of the body fluid beingtested.

The above objects as well as others are achieved by the compositions,devices and methods of this invention as disclosed herein.

SUMMARY OF THE INVENTION

The present invention provides dry chemistry reagent matrix compositioncomprising a matrix material and a reagent composition comprising3-methyl-6-(sulfonate salts)-benzothiazolinone-(2)-hydrazone (MBTH-S),N-ethyl-N-(3-sulfopropyl)aniline (ALPS), and an oxidase and/or aperoxidase enzyme, wherein the reagent composition has been imbibed anddried into or onto the matrix material. This invention is also directedto devices containing such dry chemistry reagent matrix compositions andto methods for determining the presence or concentration of analytes ina fluid sample, such as blood, using the compositions and devicesdescribed herein.

Among other factors, the present invention is based on the surprisingand unexpected discovery that a dry chemistry reagent system comprisingMBTH-S, ALPS and an oxidase enzyme and/or peroxidase enzyme permits thedetermination of the presence or concentration of an analyte in bloodwithout spectral interference from hemoglobin, i.e., it has improvedhematocrit performance. Additionally, this dry chemistry reagent systemhas a stable end point and may be formulated at a pH range of about 6.Moreover, in another embodiment of this invention, it has beendiscovered that a dry chemistry reagent system comprising MBTH-S, ALPS,N-(3-sulfopropyl)aniline (HALPS) and an oxidase enzyme and/or peroxidaseenzyme has improved dry chemistry stability compared to a dry chemistrysystem comprising MBTH-S, HALPS and an oxidase enzyme and/or peroxidaseenzyme.

Accordingly, in one of its composition aspects, the present invention isdirected to a dry chemistry reagent matrix composition comprising amatrix material and a reagent composition comprising 3-methyl-6(Msulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S) where M is apositive ion providing a stable sulfonate salt;N-ethyl-N-(3-sulfopropyl)aniline or a salt thereof; and an oxidaseenzyme or a peroxidase enzyme or mixture thereof, wherein the reagentcomposition has been imbibed and dried into or onto the matrix material.

Optionally, the reagent composition further comprises a third dyecomponent selected from the group consisting of 3,3-dimethylaminobenzoicacid, 3,5-dichloro-2-hydroxybenzenesulfonic acid,8-anilino-1-naphthelenesulfonate, N-(3-sulfopropyl)aniline or saltsthereof. Preferably, the third dye component is N-(3-sulfopropyl)aniline(HALPS) or a salt thereof.

The dry chemistry reagent composition may further comprise conventionalbinders, chelating agents, buffers and the like. Such components arewell known in the art. The dry chemistry reagent composition is imbibedand dried into or onto a matrix material (i.e., a filter material), suchas Gelman Sciences polyethersulfone membrane. The matrix materialprovides a carrier system for the reagent composition.

The MBTH-S is used in the composition in form of a stable sulfonatesalt, of which the sodium salt is preferred, but the potassium, ammoniumor other ionic form of the sulfonate salt may be used. In thisinvention, the MBTH-S is used with ALPS to form a dye couple havingimproved indication properties. Preferably, the dye composition is usedat a physiological pH, more preferably at a pH in the range of about 6to 8, still more preferably at a pH of about 6.

In another of its composition aspects, the present invention is directedto a device for testing a fluid for the presence or concentration of ananalyte comprising:

a support member comprising a matrix material;

a dry chemistry reagent composition positioned on or impregnated in saidsupport member, said reagent composition comprising 3-methyl-6(Msulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S) where M is apositive ion providing a stable sulfonate salt;N-ethyl-N-(3-sulfopropyl)aniline or a salt thereof; and an oxidaseenzyme or a peroxidase enzyme or a mixture thereof;

whereby the support member is adapted for receiving a fluid sample whichcontacts said reagent composition and adapted for the support member toprovide for inspection or reading of the color change produced by saidreagent composition after contact with the fluid sample.

The various mechanical configurations of such devices are known in theart and various configurations may be used incorporating the drychemistry reagent system of this invention adapted as desired for theparticular testing to be accomplished.

This invention also provides methods for determining the presence orconcentration of analytes in an aqueous solution, such as blood, usingthe compositions and devices described herein.

Accordingly, in one of its method aspects, this invention provides amethod of testing a fluid for the presence or concentration of ananalyte, comprising:

applying a fluid sample to a support member comprising a matrix materialhaving positioned thereon or impregnated therein a dry chemistry reagentcomposition comprising 3-methyl-6(Msulfonate)-benzothiazolinone-(2)hydrazone (MBTH-S) where M is a positiveion providing a stable sulfonate salt; N-ethyl-N-(3-sulfopropyl)anilineor a salt thereof; and an oxidase enzyme or a peroxidase enzyme or amixture thereof; whereby the support member is adapted for receiving afluid sample which contacts said reagent composition and adapted for thesupport member to provide for inspection or reading of the color changeproduced by said reagent composition after contact with the fluidsample; and

reading or measuring the color indication provided by said reagentcomposition after contact with said fluid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the reflectance at certain lightwavelengths of dye systems based on (MBTH-S)-ALPS and based onMBTH-DMAB.

FIG. 2 shows a comparison of the reflectance at certain lightwavelengths of dye systems based on (MBTH-S)-ALPS-HALPS and based onMBTH-DMAB

FIG. 3 is an illustration of the relative general activity of enzymesover a pH range.

DETAILED DESCRIPTION OF THE INVENTION

Dry chemistry indicator systems for use in test strips, such as thoseused for testing for glucose in blood, are well known in the art.Therefore, this disclosure is directed to one skilled in the art havingknowledge of how to formulate a dye or dye couple composition into a drychemistry reagent system on a test strip. The test strip typically is inthe form of an absorbent matrix adapted for containing the dry chemistryreagent indication system and for receiving the fluid sample to reactwith the dry chemistry reagent indicator system. A suitable matrix isGelman Sciences 200D polyethersulfone membrane.

Among other factors, this invention provides an improved dye indicatorsystem based on the use of 3-methyl-6-(Msulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S), wherein M is apositive ion providing a stable sulfonate sale, preferably sodium,potassium, ammonium or other equivalent ion. Dry chemistry reagent dyesystems comprising MBTH-S provide a dry chemistry system on test stripswhich are resistant to sublimation of the dye in the dry chemistrysystem and thus provide extended shelf life and increased reliability ofthe test strips containing the MBTH-S dye system. In addition, drychemistry reagent dye systems formulated based on the MBTH-S of thisinvention can be formulated and buffered to operate in a pH range ofabout 6, which provides additional stability of the oxidase enzymes orperoxidase enzymes present in the dye indicator system. In addition, thedry chemistry reagent dye systems formulated based on the MBTH-S of thisinvention also provide a stable color reaction end point which isreached in a short period of time after applying the fluid sample. Thisenables the user to read and interpret the color indication withoutdependence on accurate timing or taking readings at specific timeintervals, which usually requires the use of an electronic meter foraccurate measurement and timing. This end point stability of the dyesystem of this invention also enables the use of the test strip as atleast a semi-permanent record of the test results.

The dye systems of this invention are useful in a variety devices andsystems including those involving the reaction of whole blood or otherunfiltered fluid with the dry chemistry reagent dye system. In suchdevices and systems, the color presence of whole blood obscures tovisual inspection the indicator color change, but these systems can beread and measured by reflectance at certain specific light wavelengthsby an appropriate electronic meter system. The dye systems according tothis invention are particularly useful in devices and systems thatseparate the blood solids such as red blood cells from blood fluids andallow the clear blood fluids to contact and react with the dry chemistryreagent dye system, thus providing an unobscured, visually readablecolor change. In particular the dye system of the present invention isuseful in the devices and systems disclosed in co-pending applicationSer. No. 08/628,489 (Attorney Docket No. 018176-002) filed Apr. 5, 1996,which disclosure is incorporated herein by reference in its entirety.

In general, it will be recognized by those skilled in the art that theMBTH-S dye system of this invention can be formulated and implemented inmany of the dye systems previously based on MBTH by making theappropriate adjustment in buffer and other components to accommodate thedifferent pH range and other properties of MBTH-S compared to theconventional MBTH.

The dry chemistry reagent system of this invention is formulated toenable to analyte to react with a specific oxidase enzyme to producehydrogen peroxide which reacts with the MBTH-S based-indicator systemaccording to this invention to produce a color change which is visuallyread or electronically measured with a meter. The oxidase enzyme may beselected from glucose oxidase, cholesterol oxidase, uricase, alcoholoxidase, aldehyde oxidase, glycerophosphate oxidase or other similaroxidase enzymes known in the art to be particularly reactive with aparticular analyte. The system may also include the presence of aperoxidase enzyme such as horseradish peroxidase or other knownperoxidase to produce or enhance the desired color change in theindicator. The indicator reagent dye system is formulated in a solutionand is typically impregnated into a porous matrix or membrane such as apolyethersulfone membrane available from Gelman Science, Ann Arbor,Mich., or a fiberglass matrix available from AhlstromFiltration, Inc.,Chatanooga, Tenn., and dried to provide a dry chemistry system useful inthe conventional test strips. The MBTH-S-based dye system employed inthis invention comprises MBTH-S, N-ethyl-N-(3-sulfopropyl)aniline(ALPS), and optionally a third dye component selected from the groupconsisting of 3,3-dimethylaminobenzoic acid (DMAB),3,5-dichloro-2-hydroxybenzene-sulfonic acid(DCHBS),8-anilino-1-naphthalenesulfonate (ANS), orN-(3-sulfopropyl)aniline (HALPS). Other dye compounds which provide asufficiently high extinction point (approx. 7.0 or higher is preferred)and which form an appropriate dye couple with MBTH-S can be used in thedye system of this invention. Formulation thereof will be apparent toone skilled in the art following the teachings herein. Reference is madeto the Reagents Catalog from Dojindo Laboratories, Tokyo, Japan, and toa paper enetitled "Reagents Used for Detecting Substances in BiologicalMatrix by Enzymatic Methods" published by Dojindo Laboratories at a 1995Pacific Rim Conference, for other dyes of appropriate properties for usein this invention.

By using MBTH-S and ALPS, a dye composition is provided which can beused at physiological pH, preferably about pH 6, thereby permittingfluid samples to be tested in this pH range. Moreover, this dyecomposition has stable end point chemistry and is water soluble and doesnot sublime over time when applied and dried in the membrane matrix.Furthermore, the MBTH-S coupled with ALPS provides flat spectralabsorption in the region of about 580 to 680 nm. Moreover, at 654 nm,this system shows a good end point and minimal hematocrit interference.

It will be apparent to one skilled in the art that the selection of theadditional dye components to be combined with the MBTH-S and ALPS or toprovide dye couple systems with MBTH-S and ALPS will depend on theanalyte to be detected, the conditions under which the test strip is tobe stored and used and other conventional considerations. However it hasbeen found that the dye couple formed from the combination of MBTH-S andALPS provides a preferred dye couple system, particularly forformulation with glucose oxidase for glucose detection and measurementin blood fluids. ALPS can also be used to improve other systems, forexample, the addition of ALPS to MBTH-S/HALPS has been found to improvethe stability of that system.

The preparation of 3-methyl-6-(Msulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S) is disclosed in U.S.Pat. No. 4,101,381, issued Jul. 18, 1978, the disclosure of which isincorporated herein by reference in its entirety. The preferred MBTH-Sis where M is Na. The preferred sodium sulfonate salt is prepared asfollows: 85 grams of 3-methyl-benzothiazolinone-(2)-hydrazone (MBTH,available from Aldrich Chemical Co., Milwaukee, Wis., USA) is dissolvedin 750 grams of 25% oleum situated in an ice bath so that thetemperature is not allowed to exceed 30° C. Upon standing at roomtemperature for 12 hours complete solution is obtained. The solution ispoured into 8 liters of 0° C. water containing excess ice to maintainthe 0° C. temperature. Upon standing for about 8 hours the free sulfonicacid precipitate is filtered out, washed and dried. Approximately 85grams of crude product is obtained, which is purified by repeatextractions with boiling methanol. The resulting solid is washed withwater and dissolved in an equimolar amount of 3N NaOH with heating. Thissolution is filtered over activated charcoal and treated with a doublevolume of dioxane then allowed to crystallize overnight at about 5° C.The product is then subjected to repeated recrystallizations from 10%NaAcetate with concurrent activated charcoal filtration until thematerial is almost white. The final material is recrystallized fromwater and washed with 70% dioxane, then with pure dioxane, then withether. The resulting crystals of 3-methyl-6-(sodiumsulfonate)-benzothiazolinone-(2)-hydrazone are air dried.

N-ethyl-N-(3-sulfopropyl)aniline (ALPS) is commercially available fromDojindo Laboratories, Kumamoto, Japan.

Typically, the dry chemistry reagent system of this invention willcomprise from about 5 to about 50% by weight of MBTH-S; from about 5 toabout 50% by weight of ALPS; and from about 5 to about 50% by weight ofan oxidase or peroxidase enzyme.

The matrix material employed in the reagent test strips or devices ofthis invention are well known in the art and include, by way ofillustration, polyethersulfone, fiberglass, polyester, polyethylene, andcellulose-based membranes and the like. Such materials are commerciallyavailable from, for example, Gelman Science, Ann Arbor, Mich., USA.

The color change provide by the dry chemistry reagent system of thisinvention can be read visually or by using conventional instrumentationwell known in the art.

By way of example, a dry chemistry reagent indicator dye system isformulated as follows:

Enzyme Solution--Reagent A

The following components are combined and mixed:

30.8 mL water;

55 mL of 1 M citric acid (a buffering agent);

45 mL of 1 M triNA citrate;

20 mL of 50 mg/mL dINA EDTA;

25 mL of 100 mg/mL mannitol in dI water;

53 mL of 100 mg/mL Gantrez S95 in dI water (a color fixing agentconsisting of a polyvinyl acid, available from GAF, New York);

300 mL of 250 mg/mL Crotein SPA (a protein stabilizer consisting ofhydrolyzed collagens, available from Croda, New York);

100 mL of 11408 units/mL glucose oxidase in dI water;

100 mL of 11880 units/mL peroxidase in dI water.

Dye Solution--Reagent B

The following components are combined and mixed:

480 mL water;

500 mL ethanol;

20 mL of 100 mg/mL SOS in dI water;

6.6 g MBTH-S (sodium salt);

19.8 g ALPS.

A piece of polyethersulfone membrane from Gelman Science is uniformlycoated with Reagent A; the excess is wiped off and the membrane dried.The membrane is then coated with Reagent B in the same fashion anddried. The membrane is then assembled into a test device as shown inFIG. 2 of copending application Ser. No. 08/628,489 (Attorney Docket No.18176-002) referred to above. Whole blood is applied to the test areaand the glucose level is read by visual inspection of the colorindication on of the test side of the device. The color changes fromclear to a purple to blue color and the fmal color end point forms fromclear which is achieved in about 45 seconds and the end point color iscalibrated to known concentrations of glucose.

A second formulation was prepared using Reagent C in place of Reagent B.

Dye Solution--Reagent C

The following components are combined and mixed:

480 mL water;

500 mL ethanol;

20 mL of 100 mg/mL SOS in dI water;

6.6 g MBTH-S (sodium salt);

19.4 g HALPS;

396 mg ALPS.

This system provided the same advantages as the Reagent B-based systemand had improved dry formulation stability.

FIG. 1 shows the comparative spectral reflective in the range of 520-720nm for the dye system of the present invention based on a MBTH-S-ALPSdye couple compared to a MBTH-DMAB dye couple, such as disclosed inKiser U.S. Pat. No. 5,306,623.

FIG. 2 shows the comparative spectral reflective in the range of 520-720nm for the dye system MBTH-S-HALPS-ALPS for the dye system compared to aMBTH-DMAB dye couple.

FIG. 3 is a general illustration of the peroxidase activity of enzymesover a pH range as can be seen it is preferred to have systems which canoperate closer to a pH between about 6 and 7. The dye system accordingto the present invention is stable at a buffered pH of about 6 thusenabling the dye system of this invention to operate in a pH range morefavorable to the stability and activity of the oxidase enzymes andperoxidase enzymes present in the dye indicator system.

It will be apparent to one skilled in the art the MBTH-S-based dyeindicator of this invention can be formulated in various systems withvarious oxidase and peroxidase material to provide desired indication ofvarious analytes. It will also be apparent that the systems can beformulated at buffered pH levels which provide a favorable environmentfor stability of the enzyme components in the dry chemistry reagentsystem and in the fluid to be analyzed.

What is claimed is:
 1. A dry chemistry reagent matrix compositioncomprising a matrix material and a reagent composition comprising3-methyl-6(M sulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S) where Mis a positive ion providing a stable sulfonate salt;N-ethyl-N-(3-sulfopropyl)aniline or a salt thereof; and an oxidaseenzyme or a peroxidase enzyme or a mixture thereof, wherein the reagentcomposition is imbibed and dried into or onto the matrix material. 2.The composition according to claim 1 wherein M is a sodium, potassium orammonium ion.
 3. The composition according to claim 1 further comprisinga third dye component selected from the group consisting of3,3-methylaminobenzoic acid, 3,5-dichloro-2-hydroxybenzenesulfonic acid,8-anilino-1-naphthelenesulfonate, N-(3-sulfopropyl)aniline or saltsthereof.
 4. The composition according to claim 3 wherein the third dyecomponent is 8-anilino-1-naphthalenesulfonate or a salt thereof.
 5. Thecomposition according to claim 3 wherein the third dye component isN-(3-sulfopropyl)aniline or a salt thereof.
 6. The composition accordingto claim 1 wherein the oxidase enzyme is glucose oxidase.
 7. Thecomposition according to claim 1 wherein the matrix material is apolyethersulfone.
 8. A device for testing a fluid for the presence orconcentration of an analyte comprising:a support member comprising amatrix material; a dry chemistry reagent composition positioned on orimpregnated in said support member, said reagent composition comprising3-methyl-6(M sulfonate)-benzothiazolinone-(2)-hydrazone (MBTH-S) where Mis a positive ion providing a stable sulfonate salt;N-ethyl-N-(3-sulfopropyl)aniline or a salt thereof; and an oxidaseenzyme or a peroxidase enzyme or a mixture thereof; whereby the supportmember is adapted for receiving a fluid sample which contacts saidcomposition and adapted for the support member to provide for inspectionor reading of the color change produced by said reagent compositionafter contact with the fluid sample.
 9. The device according to claim 8wherein M is a sodium, potassium or ammonium ion.
 10. The deviceaccording to claim 8 wherein the dry chemistry reagent system furthercomprises a third dye component selected from the group consisting of3,3-dimethylaminobenzoic acid, 3,5-dichloro-2-hydroxybenzenesulfonicacid, 8-anilino-1-naphthelenesulfonate, N-(3-sulfopropyl)aniline orsalts thereof.
 11. The device according to claim 10 wherein the thirddye component is 8-anilino-1-naphthalenesulfonate or a salt thereof. 12.The device according to claim 10 wherein the third dye component isN-(3-sulfopropyl)aniline or a salt thereof.
 13. The device according toclaim 8 wherein the oxidase enzyme is glucose oxidase.
 14. The deviceaccording to claim 8 wherein the matrix material is a polyethersulfone.15. A method of testing a fluid for the presence or concentration of ananalyte comprising:applying a fluid sample to a support membercomprising a matrix material having positioned thereon or impregnatedtherein a dry chemistry reagent composition comprising 3-methyl-6(Msulfonate)-benzothiazolinone-(2)hydrazone (MBTH-S) where M is a positiveion providing a stable sulfonate salt; N-ethyl-N-(3-sulfopropyl)anilineor a salt thereof; and an oxidase enzyme or a peroxidase enzyme or amixture thereof; whereby the support member is adapted for receiving afluid sample which contacts said reagent composition and adapted for thesupport member to provide for inspection or reading of the color changeproduced by said reagent composition after contact with the fluidsample; and reading or measuring the color indication provided by saidreagent composition after contact with said fluid sample.
 16. The methodaccording to claim 15 wherein M is a sodium, potassium or ammonium ion.17. The method according to claim 15 wherein the dry chemistry reagentsystem further comprises a third dye component selected from the groupconsisting of 3,3-dimethylaminobenzoic acid,3,5-dichloro-2-hydroxybenzenesulfonic acid,8-anilino-1-naphthelenesulfonate, N-(3-sulfopropyl)aniline or saltsthereof.
 18. The method according to claim 17 wherein the third dyecomponent is 8-anilino-1-naphthalenesulfonate or a salt thereof.
 19. Themethod according to claim 17 wherein the third dye component isN-(3-sulfopropyl)aniline or a salt thereof.
 20. The method according toclaim 15 wherein the oxidase enzyme is glucose oxidase.
 21. The methodaccording to claim 15 wherein the matrix material is a polyethersulfone.